The invention described herein was developed, in part, using funds provided by the National Institutes of Health, Department of Health and Human Services, Grant No. 2-R01 DE 04321.
The detection of polynucleotides is important in many areas of molecular biology, biochemistry, biology, pharmacology and clinical medicine, inter alia. Sometimes it is important to detect the presence per se of any polynucleotide in a sample, as is the case particularly for producing protein-based therapeutic agents intended for human use, for instance. Other times it is desired to detect a specific polynucleotide sequence present in a multitude of sequences. For instance, the diagnosis of genetic disorders depends on sequence-specific polynucleotide detection and sequence-specific nucleic acid hybridization probes play a central role in molecular biology research where they are used for the detection of specific, complementary nucleic acid sequences present in minute amounts in a background of large quantities of other polynucleotides. Assays involving nucleic acid hybridization in the future will be ubiquitous in the clinical lab, and will find widespread application in other areas, including, for instance, veterinary medicine, forensic medicine, plant breeding and other areas of agriculture.
Whether it is desired to detect the presence, per se, of polynucleotides in sample, or to detect a particular polynucleotide sequence, a key step is labeling the polynucleotide or polynucleotide probe with a detectable label. Generally, the specificity and sensitivity of a detection method will be at least partly determined by the properties of the detectable label and the efficiency of the labeling method used to introduce the label into the polynucleotide.
Various labeling techniques have been developed that provide the selectivity and efficiency necessary to detect DNA present at fairly low levels in a sample, and to label hybridization probes sufficiently to detect specific sequences of DNA present in a high background of other sequences. For instance, radioisotopes such as .sup.3 H, .sup.4 C, .sup.32 p, .sup.35 S and .sup.125 I have been incorporated into polynucleotides by metabolic, enzymatic and chemical means to serve as detectable reporter groups to indicate the presence of polynucleotides in a sample. .sup.32 p and .sup.35 S, have proven particularly useful in molecular biology studies in such techniques as RNA and DNA sequencing, and the panoply of blot-hybridization methods used to detect, identify, measure, quantitate and localize specific polynucleotide sequences.
Radioactive labels suffer from several drawbacks, however. First, there is the risk of human exposure to hazardous levels of radioactivity during the preparation, use and disposal of reagents containing a radioactive tag. In consequence of the risk associated with human exposure to radiation there is a need when working with radiation for elaborate and expensive safety precautions.
In addition, the radioisotopes most suitable for use in nucleic acid research have relatively short half-lives. For instance, .sup.32 p has a half-life of only 14 days, and .sup.35 S has a half-life of only 87 days. Radioactively labeled probes therefore have limited shelf-lives and cannot be prepared and standardized in large batches, well before actual use. The necessity to prepare probes in small batches close to the time of actual use incurs economic disadvantages of scale. Furthermore, the inability to prepare and characterize large batches of radioactively labeled probes is a barrier to developing polynucleotide based diagnostic reagents.
The disadvantages of radioactive labels has led to the development of alternative techniques for introducing stable non-radioactive, detectable labels into polynucleotides. Thus, methods have been developed to label polynucleotides with, inter alia, biotin, digoxigenin and sulfonate.
Perhaps the most widely used non-radioactive polynucleotide label is biotin. Biotin binds with exceptional specificity and avidity to the proteins avidin and streptavidin. Thus, these proteins will bind with exceptional efficiency to biotinylated DNA. Both proteins can be cross-linked to an enzyme to form an enzymatically active conjugate, in which the avidin or streptavidin portion specifically detects the biotin in biotinylated DNA and the enzymatic portion catalyses the formation of a detectable product, such as a colored or luminescent product, which can be determined quantitatively and indicates the presence and the amount of biotinylated DNA in the sample. Avidin-alkaline phosphatase conjugates which are compatible with standard EIA colorimetric reagents have been widely employed in this type of biotin-based procedure.
Other labels, such as digoxigenin, can be used in analogous fashion. Thus, digoxigenin may be incorporated into the DNA analogously to biotin and the digoxigenin-DNA adduct detected by ELISA or other EIA techniques using an enzyme conjugated to an anti-digoxigenin polyclonal or monoclonal antibody.
In view of the widespread utility of labeled polynucleotides, simple, efficient and reliable methods for labeling polynucleotides are needed. The methods presently available, however, suffer from a number of disadvantages. For instance, biotinylated polynucleotides are produced using biotinylated nucleoside derivatives which must be chemically synthesized and then incorporated into a polynucleotide by enzymatic or chemical reactions. Thus, substrates for the preparation of biotinylated and digoxigeninylated polynucleotides are analogues of the native nucleoside and deoxynucleoside triphosphates, with chemical groups covalently coupled to the base. Examples of such analogues are uridine and deoxyuridine triphosphate (UTP and dUTP) coupled via a spacer arm to biotin (biotin-11-dUTP and biotin-11-UTP), or to digoxigenin (digoxigenin-11-dUTP and digoxigenin-11-UTP), in which the detectable group is attached at C5 of the uridine base. See Langer et al., Proc. Nat'l. Acad. Sci., U.S.A. 78: 6633-6637 (1981) and Brigati et al., Virology 126:36-50 (1983).
Methods for synthesizing biotinylated or digoxigeninylated precursors, however, are time-consuming and require considerable expertise in synthetic chemistry. Moreover, labeling polynucleotides with these substrates generally requires expensive enzymes and exacting reaction conditions. Thus, such substrates cannot be used in many situations and are not suitable for the synthesis of large amounts of labeled polynucleotide.
Photoreactive derivatives of biotin and digoxigenin, such as those described by Forster et al., Nucl. Acids Res. 13: 745 (1985), provide a non-enzymatic means of incorporating biotin or digoxigenin into polynucleotides, including double-stranded DNA (dsDNA), single-stranded DNA (ssDNA) and RNA. The photoreactive derivatives must be chemically synthesized, however, a technically demanding and expensive process. In addition, the polynucleotides being labeled must be pure since the photochemical reactions are not specific and contaminating compounds such as proteins are readily labeled. The density of labeling is low with this procedure, being on the order of one label per 200 base residues. Furthermore, polynucleotides labeled using the photoreactive derivatives often bear biotin or digoxigenin on sites involved in Watson-Crick hydrogen bonding, which can deleteriously affect the ability of a probe to bind to its target sequence in a sample.
Several chemical procedures for incorporating detectable groups into polynucleotides have been described. For instance, Stavrianopoulos in U.S. Pat. No. 4,843,122 described a method for biotinylating DNA wherein guanine C8 is activated with 3,4,5-trichloro-aniline and then bonded to biotin-SH. Reisfeld et al., Biochem. Biophys. Res. Comm. 142:519 (1987), described the bisulfite catalyzed bonding of biotin hydrazide to cytidine N.sup.4 in DNA. Takahashi et al., Nucleic Acids Res. 17: 4899 (1989), described the coupling of biotin aminocaproylhydrazide to single-stranded DNA by reaction with glutaraldehyde, possibly resulting in substitution at N.sup.6 of adenosine, N.sup.2 of guanosine and N.sup.4 of cytosine. Sverdlov et al., Biochim. Biophys. Acta. 340:153 (1974), described the sulfonation of cytosine C6, accompanied by substitution of the exocyclic N.sup.4 amino group with methoxyamine, forming N.sup.4 -methoxy-5,6-dihydro-cytosine-6-sulfonate.
Biotin ligands in polynucleotides labeled according to these procedures serve as biotin receptor (e.g., avidin and streptavidin) binding sites, allowing detection of biotinylated DNA by ELISA and other EIA methods, inter alia, as described above. Similarly, polynucleotides labeled by N.sup.4 -methoxy-5,6-dihydro-cytosine-6-sulfonate bind a monoclonal antibody against N.sup.4 -methoxy-5,6-dihydro-cytosine-6-sulfonate and are detected in much the same way as biotin by ELISA and other EIA methods.
All of these methods suffer from a variety of disadvantages. Some methods require cumbersome procedures. Some methods can introduce only a single molecule of detectable label into each polynucleotide molecule. None of the methods can be used to label polynucleotides in a complex mixture of many different contaminants.
Methods have also been described for incorporating a label into a polynucleotide made by chemical synthesis techniques. For instance, Ruth, DNA 3: 123 (1984), described a method for incorporating detectable labels into purified polynucleotides of defined sequence by chemical synthesis. Ruth described nucleosides bearing functionalized linkers suitable for use in oligonucleotide synthesizers. Synthetic single-stranded oligonucleotides of defined sequence were produced bearing the functionalized linkers on pre-selected bases in the sequence. The functional group could be a detectable label or could be joined to a detectable label following synthesis. Production of the derivatized nucleosides of Ruth's method requires considerable technical skill, however, and their utility is limited to labeling synthetic oligonucleotides.
Jablonski et al., Nucleic Acids Res. 14:6115 (1986), showed that the homobifunctional reagent disuccinimidyl suberate can be used to directly cross-link an oligonucleotide with the enzyme alkaline phosphatase, through a single amine-modified base in the oligonucleotide. This method, however, is limited to oligonucleotides containing the amine-modified base. Furthermore, the alkaline phosphatase may interfere with base pairing by the oligonucleotide in a hybridization assay.
In sum, despite the importance of labeled polynucleotides in molecular biology, biochemistry and clinical diagnostic applications, inter alia, a simple, reliable, efficient and widely applicable procedure for introducing detectable labels into polynucleotides has not been developed. All of the procedures discussed hereinabove are of limited applicability. Few methods provide for introducing many labels into each polynucleotide. Conventional enzymatic labeling methods require expensive enzymes, highly purified nucleic acid templates and substrates, and reaction conditions must be stringently controlled. Furthermore, the efficiency of labeling by these methods is difficult to determine, making it hard to assess usefulness of a labeled product before use.
Photochemical and chemical (sulfonation) procedures have a wider range of applicability but require cumbersome chemical procedures and also require purified nucleic acid substrates. Although many of the prior art procedures can be completed within a few hours, they may require extensive preliminary preparations and the entire process is usually very time consuming. Furthermore, excepting photochemical labeling and the automated synthesis of labeled oligonucleotides, labeling procedures yield very small amounts (microgram quantities) of labeled product.